Borrelia spirochetes are responsible for a variety of human disorders including Lyme borreliosis and relapsing fevers. Lyme disease is an infection caused by the spirochete, B. burgdorferi, which is carried by ticks. The spirochete is transmitted to humans and animals through the bite of a tick and can cause serious dermatological, arthritic, neurological and other pathological disorders in an infected host. Recently, Lyme disease has become a serious epidemiological concern in North America, as well as Europe, Asia and the Soviet Union.
Relapsing fevers are also caused by Borrelia spirochetes and are either tick-borne or louse-borne. Louse-borne relapsing fever is caused by the Borrelia strain B. recurrentis, while tick-borne relapsing fevers are caused by any one of a number of Borrelia strains including, in the United States, B. hermsi, B. parkeri and B. turicatae.
It is well documented that persons and animals infected by Borrelia pathogens typically develop antibodies in response to the presence of various Borrelia antigens, including outer membrane lipoproteins. For example, patients infected with Lyme disease develop antibodies to outer surface protein A (OspA), a lipoprotein of the B. burgdorferi spirochete. See Craft, J. E., Fischer, D. K., Shimamoto, G. T., and Steere, A. C., "Antigens of Borrelia burgdorferi recognized during Lyme disease. Appearance of a new immunoglobulin in response and expansion of the immunoglobulin G response late in the illness, " J. Clin. Invest., 78:934-939 (1986). See also, Barbour, A. G., Heiland, R. A., and Howe, T. R., "Heterogeneity of major proteins in Lyme disease borrelia: a molecular analysis of North American and European isolates," J. Infect. Dis., 152:478-484 (1985) The outer surface protein A (OspA) is a lipoprotein encoded by the nucleotide sequence of the ospA gene present in the DNA of the B. burgdorferi spirochete. The nucleotide sequence coding for the full-length, wild-type OspA (see SEQ ID NO: 1) has been previously determined for B31, the North American strain of B. burgdorferi. See Bergstr om, S., Bundoc, V. G., & Barbour, A. G., "Molecular Analysis of linear plasmid-encoded major surface proteins, OspA and OspB of the Lyme disease spirochete Borrelia burgdorferi," Mol. Microbiol., 3:479-486 (1989). Consequently, the OspA amino acid sequence has been predicted from the nucleotide data (see SEQ ID NO: 2).
Relapsing fever is characterized by repeated episodes of illness and fever separated by periods of well being (see, for example, N. Burman et al., Mol. Micro. 4(10):1715-1726 (1990)). The relapsing fever borreliae have developed a mechanism of multiphasic antigenic variation to avoid the immune response of mammalian hosts. The antigenic switch in B. hermsii is associated with an outer membrane protein designated `variable major protein`or Vmp. Vmps of a number of different B. hermsii serotypes have been shown to exhibit amino acid sequence variability in many regions of the protein. This sequence variability, or multiphasic antigenic variation, is responsible for the organism's ability to repeatedly escape from the infected host's immune surveillance.
From a clinical standpoint, it is highly desirable to develop a method of producing large quantities of highly purified Borrelia lipoproteins in a soluble form for use in immunoassays and other diagnostic screening tests which detect the presence of antibodies to these proteins in the sera of patients infected with Borrelia spirochetes. Furthermore, soluble, highly purified forms of these lipoproteins would be potentially valuable as clinical immunogens for vaccinating both people and animals against Borrelia pathogens, as well as useful research tools for subsequent laboratory manipulations involving the separation and purification of antibodies to such proteins.
To this end, it is highly desirable to obtain a nucleotide sequence or gene which can be expressed at high levels in a recombinant host/vector expression system to yield large quantities of the resulting recombinant protein while retaining the desired specific reactivity.
Previous attempts have been made to isolate purified, soluble Borrelia lipoproteins through the growth and subsequent purification of Borrelia cell cultures. There are several drawbacks to this approach, however. The growth and subsequent purification of these proteins from crude cell extracts of Borrelia is very time consuming and expensive. Additionally, the growth and manipulation of live Borrelia cultures adds significant risk to laboratory personnel. Most importantly, the full-length, wild-type versions of Borrelia lipoproteins yielded by this method have poor solubility properties because these proteins have a hydrophobic, lipidated character due to their post-translational covalent modification which takes place at the cell membrane of the spirochete during expression. Consequently, detergents are required to solubilize these lipidated proteins.
It is well accepted in the art that the treatment of lipoproteins with detergents improves solubility but often impairs reactivity by altering or destroying the folding configuration of the target protein as well as the epitopic sites. Consequently, it would be desirable to develop a recombinant variation of OspA as well as other Borrelia lipoproteins that are soluble without exposure to detergents while retaining specific reactivity to antibodies against their full-length, wild-type lipoprotein analogs. In addition to the foregoing solubility problems, the association of the Borrelia lipoproteins with the cell membrane of the spirochete also creates problems in the separation and purification of these proteins from crude cell extracts.
As an alternative approach to the production of Borrelia lipoproteins, certain recombinant DNA techniques can be utilized to express Borrelia genes using a host/vector expression system such as Escherichia coli containing recombinant cloning vectors known in the art. A suitable recombinant cloning vector would be a plasmid having a nucleotide sequence that could be modified to accept an insertion of wild-type Borrelia DNA. While these recombinant techniques avoid the need for live Borrelia cultures, they have several shortcomings.
For example, recombinant versions of the full-length, wild-type Borrelia lipoproteins produced in E. coli have poor solubility properties in the absence of detergents, presumably due to post translational modification of the protein at the cell membrane of the host during expression. Consequently, subsequent manipulations directed to the separation and purification of the resulting protein product involve problems similar to those encountered when attempting to isolate and purify OspA from live B. burgdorferi cultures.
Another shortcoming of the above approach is that recombinant versions of the full-length, wild-type ospA and ospB genes are expressed at very low levels in an E. coli host. This poor expression is presumably due to the accumulated toxic effects of Borrelia lipoprotein localization at the E. coli cell membrane during the course of expression.